Note – the shortage of competent cells is not as severe
as was reported

In this lab, a 5.3 kb PstI fragment will be purified
from an agarose gel, subcloned into PstI-cut pUC19, and transformed into
DH5α (DH5alpha) competent cells.

Elution of gel band

Before the lab, plasmid pPBH (pBluescript containing a
fragment of P. ochraceus histone gene sequence) will be digested with PstI.0.5 µg aliquots of the digested DNA will be
loaded on agarose gels, along with a 1 Kb Ladder molecular weight marker.After the gels have run long enough for the
bromophenol blue band to be near the end of the gel, two bands should be
visible in the sample lanes:a 5.3 kb
histone gene fragment, and a 3 kb vector fragment.The 5.3 kb histone gene band will be cut out of the gel, and
eluted using the GFX PCR DNA and Gel Band Purification Kit.This is a kit which uses NaI to dissolve
agarose and a silica matrix column to bind the eluted DNA.For instructions on how to use the kit, see
the “Subcloning lab references” on reserve in the library.The most important step in using the kit
properly is to cut out the gel band in the minimum amount of agarose
possible.This will reduce the amount of
the “capture buffer” needed to completely digest the agarose.Also be careful not to expose the DNA to UV
light for more time than is necessary – this will minimize DNA damage due to UV
radiation.

Assuming complete recovery of the band, the amount of DNA
recovered should be a fraction of the 0.5 µg of DNA loaded on the gel
proportional to the percent that the 5.3 kb fragment made up of the original
8.3 kb plasmid.(E.g. 0.5 µg X 5.3
kb/8.3 kb).The
fragment will be eluted from the columns using 50 µl sterile ddH2O.(TE could also be used for this purpose, but
the EDTA might interfere with the ligation step.T4 DNA ligase requires Mg2+ as a cofactor.)

Protocol:

1.When bromophenol blue band is near
end of gel, remove gel from apparatus and place on saran wrap.Photograph the gel, then take it to the
darkroom in lab 8159.

2.Follow the protocol for the GFX kit
(see the “Subcloning lab references” on reserve in the library).

3.Elute the DNA using 50 µl sterile ddH2O.Assuming complete recovery of the band,
calculate the concentration of the DNA present.

Ligation

For maximum yield, ligations are usually done at 14-16°C
overnight.(1-4 hours at room
temperature will also give decent yields.)Because of time constraints, the ligation in this lab will be done using
a “Rapid Ligation” protocol suggested by Invitrogen/Life Technologies, which is
supposed to produce results in 5 minutes at room temperature.The main difference between this and the
standard ligation protocol is the higher concentration of ligase used – 1 Weiss
unit per reaction, instead of the 0.1 unit per reaction that is normally used
for cohesive end ligations.(See the “Subcloning
lab references” on reserve in the library).To increase the chances of ligation between vector and insert, a
ratio of 3 insert DNA ends to 1 vector DNA end will be used.Since ligase joins DNA ends together, the
concentrations of DNA ends (in fmols) for both DNA molecules must be known.

fmols DNA ends can be calculated using the following
formula:

1 µg DNA (for a 1 kb fragment) = 3000 fmol DNA ends

µg DNA = fmol DNA ends X 1 µg/3000 fmol
X size of DNA in kb/1 kb

(See the “Subcloning lab references” on reserve in
the library for examples of calculations).

Two ligations will be set up by each group – a test ligation
containing insert and vector DNA, and a control ligation containing only vector
DNA.Because T4 DNA ligase is not
stable for long at 0°C, the 1 unit/µl ligase working stock will be prepared
each day shortly before use.T4 DNA
ligase buffer contains ATP, so it must be kept on ice while being used.The polyethylene glycol (PEG) in Life
Technologies DNA ligase buffer may precipitate during freezing – therefore
thawed buffer should be vortexed vigorously before use.

If you wish to see recipes for making competent cells, consult
the Sambrook reference in the lab.

Each group will do 5 transformations:the test ligation and 4 controls.The controls will be the control ligation,
unligated PstI-cut pUC19 vector DNA, pPBH plasmid miniprep (from Lab 3),
and a mock transformation with no DNA added.Different amounts of DNA are needed for the different reactions, because
supercoiled plasmid DNA transforms competent E. coli cells more
efficiently than open-circle ligated circular plasmid DNA.Linear DNA transforms chemically competent E.
coli at a very low rate.

Materials (per group):

7 plates (LB + 100 µg/ml ampicillin)

X-gal (20 mg/ml)

IPTG (20% w/v)

1 X 125 µl aliquot of competent cells

diluted test ligation (100 µl)

diluted control ligation (100 µl)

unligated PstI-cut pUC19

pPBH plasmid miniprep (from Lab 3)

1.Aliquot 1 X 125 µl aliquot of
competent cells from one of the stock tubes.Divide this into 5 X 25 µl of competent cells on ice in 5 tubes.

2.Set up the following transformations.Tubes should be kept on ice while you set up the mixes.

Ingredient

Test ligation

Control ligation

Unligated digested pUC19

pPBH plasmid miniprep

Mock ligation

Competent cells

25 µl

25 µl

25 µl

25 µl

25 µl

DNA

10

10 µl

2 µl

~0.5 µl

none

3.Leave
transformation mixes on ice for 10 minutes.

4. Swirl the
tubes in the 37°C waterbath for 2-3 minutes.Return to ice.